Relative contributions of dectin-1 and complement to immune responses to particulate β-glucans.

Abstract

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.

Effect of Dectin-1 and complement on phagocytosis of GPs by BMDCs in vitro

Wild-type (A and C) or Dectin-1−/− BMDCs (B) were incubated with F-GPs without opsonization (No MS) or opsonized with fresh mouse serum (MS) or heat-inactivated mouse serum (MS HI). Wild-type mouse serum was used in panels A and B and C3−/− mouse serum was used in panel C. Laminarin (Lam, 1 mg/ml [final concentration]) was added to wells where indicated. The percentage of F-GP+ BMDCs with one or more F-GP particle(s) per cell was determined by epifluorescent microscopy. Results expressed as mean ± SE of at least two independent experiments, each performed in duplicate or triplicate. **: p < 0.01, ***: p < 0.001 comparing with or without laminarin.

Involvement of CR3 in the phagocytosis of GPs by bone marrow-derived dendritic cells (BMDCs) in vitro

Wild-type BMDCs were pre-incubated with α-CR3 mAbs (a mixture of M1/70 and 5C6) or a control rat IgG2b mAb for 1 h before incubation with F-GPs. F-GPs were not incubated with mouse serum (A), opsonized with fresh wild-type mouse serum (B) or incubated with heat-inactivated wild-type mouse serum (C). Laminarin (Lam, 1 mg/ml final concentration) was added to wells 1 h before F-GPs, where indicated. Quantification of F-GP phagocytosis was accomplished as in Figure 1. Results represent mean ± SE of one experiment with 3 determinations (A and C) and two independent experiments, each with 3 determinations (B). **: p< 0.01 comparing α-CR3 with No Abs or IgG2b; ***: p < 0.001 comparing α-CR3 with No Abs or IgG2b.